Multiplex detection methods, antigen screening methods and kits for infertility autoantibodies

Abstract

This invention discloses a multiplex detection method, antigen screening method, and kit for infertility-related autoantibodies. The detection method includes: conjugating antigens corresponding to different infertility-related autoantibodies to different solid-phase carriers and blocking them; labeling secondary antibodies with phycoerythrin; mixing the analyte with a capture carrier and a secondary antibody-phycoerythrin complex to form an immune complex; after excitation by a light source, determining the type of analyte based on the fluorescence signal of different solid-phase carriers, and calculating the concentration based on the phycoerythrin fluorescence signal. The antigen screening method uses a relative deviation of ±10% as a standard to screen antigen combinations without cross-interference from multiple candidate antigens. The kit contains six antigen-conjugated solid-phase carriers and a secondary antibody-phycoerythrin complex. This invention enables simultaneous detection of six infertility-related autoantibodies with a single sample addition and low sample volume. The correlation coefficient (r) between the detected values ​​and theoretical values ​​of each item is >0.99, making it suitable for auxiliary medical diagnosis of infertility.

Description

Technical Field

[0001] This invention relates to the field of medical diagnostics, specifically to a method for multiplex detection of autoantibodies in infertility, an antigen screening method, and a reagent kit. Background Technology

[0002] Infertility is a common reproductive system disease with a high incidence rate, seriously impacting the reproductive health and quality of life of modern women. Infertility primarily refers to the inability of a woman to conceive despite regular sexual intercourse and the use of contraception. Female infertility is closely related to certain autoantibodies, which can negatively affect egg formation and development, the union of sperm and egg, fertilization, endometrial receptivity, implantation, embryonic development, and maternal immune response to the fetus.

[0003] Infertility-related autoantibodies (including antisperm antibodies (AsAb), anti-endometrial antibodies (EMAb), anti-ovarian antibodies (AoAb), anti-zona pellucida antibodies (AZPAb), anti-human chorionic gonadotropin antibodies (AHCGAb), and anti-trophoblast cell antibodies (TaAb)) play an important role in the auxiliary medical diagnosis of female infertility. Literature indicates that the positive rates of antisperm antibodies (AsAb), anti-endometrial antibodies (EMAb), anti-ovarian antibodies (AoAb), and anti-zona pellucida antibodies (AZPAb) in the serum of infertile women were significantly higher than those in the control group, with statistically significant differences (P<0.01). The positive rates of anti-human chorionic gonadotropin antibodies in the serum of patients with secondary infertility and those with primary and secondary infertility were significantly higher than those in the normal reproductive-age group and the primary infertility group, with statistically significant differences (P<0.0001). The positive rates of anti-trophoblast cell antibodies in the serum of women with miscarriage were also significantly higher. The levels of anti-trophoblast antibodies in women with polycystic ovary syndrome (PCOS) were significantly higher than those in women with normal pregnancies (P < 0.001). The levels of serum anti-trophoblast antibodies in women with miscarriage were positively correlated with parity (r = 0.91, P < 0.05). The levels of serum anti-sperm antibodies, anti-endometrial antibodies, and anti-ovarian antibodies in women with poor pregnancy outcomes were significantly higher than those in women with good pregnancy outcomes (P < 0.05). The levels of serum anti-sperm antibodies, anti-endometrial antibodies, and anti-ovarian antibodies were positively correlated with the risk of poor pregnancy outcomes in women with PCOS (P < 0.05).

[0004] Immunoassays in medical diagnostics utilize highly specific antigen-antibody binding reactions to detect and analyze specific substances in biological samples. Currently, the primary immunoassay method for detecting infertility-related autoantibodies is enzyme-linked immunosorbent assay (ELISA). This method employs an indirect approach, binding an antigen to a solid, which then forms an antigen-antibody-enzyme-labeled secondary antibody immune complex with the analyte and enzyme-labeled secondary antibody in the sample. Detection is then performed via enzymatic substrate catalysis. The methods mentioned in the aforementioned literature for detecting infertility-related autoantibodies are all ELISA methods. Due to its advantages such as mature carriers, simple procedures, and low instrument dependence, ELISA has experienced rapid development and widespread application. Although ELISA is relatively mature, it is still commonly used to measure single analytes, has a long reaction time, requires large sample volumes, and its precision and detectable range no longer meet current medical diagnostic needs. For situations where samples are difficult to obtain, such as difficulties in sampling specific populations or limited sample volumes for specific sample types, rapid multiplex detection techniques that allow for single sample loading and low sample volume requirements remain a challenge for immunoassays.

[0005] Therefore, rapid multiplex detection methods that can jointly detect infertility-related autoantibodies have high application value in assisting clinicians in providing fertility guidance, assessing the pregnancy risk of infertile patients, and providing corresponding treatment measures. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for multiplex detection of autoantibodies in infertility, an antigen screening method, and a kit, thereby solving the problems of rapid multiplex detection with low sample volume requirements that cannot be achieved with single sample loading in existing technologies. At the same time, it also solves the problem that cross-interference between different autoantibodies due to similar structures and specific binding sites can easily affect the accuracy of detection.

[0007] The first technical solution adopted in this invention is a method for multiplex detection of infertility autoantibodies, comprising the following steps: conjugating antigens corresponding to different infertility-related autoantibodies to different solid-phase carriers; blocking the solid-phase carriers to form a capture carrier; labeling phycoerythrin onto a secondary antibody to form a secondary antibody-phycoerythrin complex; mixing the analyte with the capture carrier and the secondary antibody-phycoerythrin complex to form a capture carrier-analyte-secondary antibody-phycoerythrin immune complex; exciting the immune complex with a light source to generate a corresponding fluorescence signal; determining the type of analyte and calculating the concentration of the analyte based on the different fluorescence signals.

[0008] Preferably, different solid-phase supports produce different fluorescence signals when excited by a light source, and the fluorescence signal of the phycoerythrin is related to the concentration of the analyte.

[0009] Preferably, the solid support is a magnetic microsphere.

[0010] Preferably, the infertility-related autoantibodies are selected from at least two of the following: antisperm antibodies, antiendometrial antibodies, antiovarian antibodies, antizona pellucida antibodies, antihuman chorionic gonadotropin antibodies, and antitrophoblast cell antibodies.

[0011] The second technical solution adopted in this invention is an antigen screening method for multiplex detection of infertility autoantibodies, comprising the following steps: providing multiple sets of candidate antigens, each set containing antigens corresponding to different infertility-related autoantibodies; using each candidate antigen set to perform multiplex detection on interfering samples containing a single infertility-related autoantibody that is positive; using a relative deviation of ±10% in the detection results of each item as the criterion for no interference; and screening out antigen sets that do not interfere with the detection of all interfering samples.

[0012] Preferably, the infertility-related autoantibodies include antisperm antibodies, anti-endometrial antibodies, anti-ovarian antibodies, anti-zona pellucida antibodies, anti-human chorionic gonadotropin antibodies, and anti-trophoblast cell antibodies.

[0013] The third technical solution adopted in this invention is: an infertility autoantibody detection kit, comprising the following components: a solid-phase carrier 1 conjugated with antisperm antibody antigen, a solid-phase carrier 2 conjugated with anti-endometrial antibody antigen, a solid-phase carrier 3 conjugated with anti-ovarian antibody antigen, a solid-phase carrier 4 conjugated with anti-zona pellucida antibody antigen, a solid-phase carrier 5 conjugated with anti-human chorionic gonadotropin antibody antigen, a solid-phase carrier 6 conjugated with anti-trophoblast cell antibody antigen; and a secondary antibody-phycoerythrin complex.

[0014] Preferably, the solid support is a magnetic microsphere.

[0015] Preferably, the antigens are: antisperm antibody antigen NDX-AsAb01, anti-endometrial antibody antigen NDX-EMAb01, anti-ovarian antibody antigen NDX-AoAb01, anti-zona pellucida antibody antigen NDX-AZPAb01, anti-human chorionic gonadotropin antibody antigen NDX-AHCGAb01, and anti-trophoblast cell antibody antigen NDX-TaAb01.

[0016] The beneficial effects of this invention are as follows: 1. Achieving simultaneous multiplex detection with a single sample loading and low sample volume: This invention utilizes solid-phase carriers with different fluorescent codes. Different analytes in the biological sample react with their corresponding solid-phase carriers and antibodies in a one-step reaction to form immune complexes. Upon excitation by a light source, these complexes generate fluorescence signals. The type of analyte can be determined by the fluorescence signals of different solid-phase carriers, and the concentration of the analyte can be calculated using the fluorescence signal of phycoerythrin. Therefore, this invention enables multiplex detection with a single sample loading and low sample volume requirements. A single sample loading can simultaneously obtain the detection results of six infertility-related autoantibodies.

[0017] 2. Systematic antigen screening effectively eliminates cross-interference and ensures detection specificity: Autoantibodies with similar structures and specific binding sites are prone to cross-reactions, affecting the accuracy of multiplex detection. This invention addresses this technical challenge by proposing a systematic antigen screening method. By comparing and analyzing multiple antigen sets, using a relative deviation of ±10% as the criterion, cross-reactions and interference reactions between different autoantibody items are effectively eliminated. Experimental results show that the relative deviations of the screened antigen sets for the six single-positive interfering samples are all within ±10%, with no endogenous interference. In contrast, other unscreened antigen sets showed deviations exceeding ±10%, demonstrating the effectiveness of the screening method of this invention.

[0018] 3. Detection performance meets medical diagnostic standards: The detection results of this invention on a composite gradient of six infertility-related autoantibodies showed that the correlation coefficients (r values) between the detected values ​​of antisperm antibody, anti-endometrial antibody, anti-ovarian antibody, anti-zona pellucida antibody, anti-human chorionic gonadotropin antibody, and anti-trophoblast cell antibody and the theoretical values ​​of the samples were 0.999, 0.999, 0.999, 0.998, 0.999, and 0.998, respectively, all greater than 0.99, meeting the requirements of generally accepted medical diagnostic standards.

[0019] 4. High degree of commercialization, enabling fully automated detection: The raw materials used in this invention are all commercially available and mass-produced, with no special modification requirements. The experimental design of this invention is simple, requiring no complex or sophisticated equipment to complete item identification and concentration detection. The detection and analysis process can be fully automated, requiring only a single sample addition reaction for each sample, eliminating the need for multiple additions and repeated reactions, thus improving the clinical application efficiency of infertility-related autoantibody detection and reducing testing costs. Attached Figure Description

[0020] Figure 1 This is a flowchart of the multiplex detection method for infertility autoantibodies of the present invention. Detailed Implementation

[0021] The following examples further illustrate the present invention, but are not intended to limit the invention. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments with the same designations. However, any modifications, equivalent changes, or alterations made to the following embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

[0022] I. Overall Procedure for Multiplex Detection of Autoantibodies in Infertility like Figure 1 As shown, the multiplex detection method for infertility autoantibodies provided in this embodiment includes the following steps: Antigens corresponding to different infertility-related autoantibodies are coupled to different solid-phase carriers; The solid-phase support is sealed to form a capture carrier; Phycoerythrin was labeled onto the secondary antibody to form a secondary antibody-phycoerythrin complex; The analyte is mixed with the capture carrier and the secondary antibody-phycoerythrin complex to form a capture carrier-analyte-secondary antibody-phycoerythrin immune complex. The immune complex is excited by a light source to generate a corresponding fluorescence signal; The type of analyte is determined and its concentration is calculated based on the different fluorescence signals.

[0023] Understandably, when testing different analytes in a single sample with a single addition, different antigens corresponding to the analytes need to be used based on the specificity of the binding between the analyte and the antigen. For example, when testing for six analytes (infertility-related autoantibodies 1-6) in the same sample, the antigens corresponding to these six analytes (antigens 1-6) need to be used to specifically bind with the analytes in the sample, forming antigen-analyte complexes. A universal anti-human antibody is selected as the secondary antibody to form the antigen-analyte-secondary antibody complex.

[0024] This embodiment employs different solid-phase carriers, each generating different fluorescence signals upon excitation by a light source. When different analytes in the same sample specifically bind to the antigen on the surface of their respective capture carriers and the secondary antibody in the secondary antibody-phycoerythrin complex, the light source excites all the capture carrier-analyte-secondary antibody-phycoerythrin immune complexes to produce fluorescence signals. The different fluorescence signals from the solid-phase carriers can distinguish the types of analytes, and the concentration of the analyte can be calculated using the fluorescence signal of phycoerythrin. Therefore, rapid, multiple infertility-related autoantibody detection can be achieved for a single sample with a single sample loading.

[0025] Based on the above method, this embodiment provides an infertility autoantibody detection kit, comprising the following components: a solid-phase carrier 1 conjugated with antisperm antibody antigen, a solid-phase carrier 2 conjugated with anti-endometrial antibody antigen, a solid-phase carrier 3 conjugated with anti-ovarian antibody antigen, a solid-phase carrier 4 conjugated with anti-zona pellucida antibody antigen, a solid-phase carrier 5 conjugated with anti-human chorionic gonadotropin antibody antigen, a solid-phase carrier 6 conjugated with anti-trophoblast cell antibody antigen; and a secondary antibody-phycoerythrin complex.

[0026] II. Antigen Screening When establishing a multiplex detection system, cross-reactions may occur between autoantibodies with similar structures and specific binding sites. This can manifest as cross-reactions or interference reactions; for example, a particular autoantibody may react with the antigens of other autoantibodies, leading to elevated test results for those other autoantibodies. To eliminate this interference, this embodiment collects and screens information on commercially available mass-produced raw materials, selecting three antigen sets for comparative analysis. The information for the three antigen sets is as follows.

[0027] Set 1: Antisperm antibody antigen a, anti-endometrial antibody antigen a, anti-ovarian antibody antigen a, anti-zona pellucida antibody antigen a, anti-human chorionic gonadotropin antibody antigen a, anti-trophoblast cell antibody antigen a.

[0028] Set 2: Antisperm antibody antigen b, anti-endometrial antibody antigen b, anti-ovarian antibody antigen b, anti-zona pellucida antibody antigen b, anti-human chorionic gonadotropin antibody antigen b, and anti-trophoblast cell antibody antigen b.

[0029] Set 3: Antisperm antibody antigen c, anti-endometrial antibody antigen c, anti-ovarian antibody antigen c, anti-zona pellucida antibody antigen c, anti-human chorionic gonadotropin antibody antigen c, anti-trophoblast cell antibody antigen c.

[0030] Antigens from kits 1, 2, and 3 were prepared according to the reagent preparation methods described in the examples below, resulting in reagent kits 1, 2, and 3, respectively. Six interfering samples were then tested using each of the three kits.

[0031] The information for the six interfering samples is as follows (RU / mL):

[0032] Six samples each tested positive for one item and negative for the others. The relative deviation of each item in the six samples should be within ±10%. If the relative deviation exceeds ±10%, it indicates that the positive sample for the corresponding item interfered with the detection of other items.

[0033] 1. Test results and interference analysis of reagent group 1 Test results (RU / mL):

[0034] Relative deviation:

[0035] Interference analysis: Positive samples for anti-endometrial antibodies interfered with the detection of anti-ovarian antibodies and anti-zona pellucida antibodies; positive samples for anti-ovarian antibodies interfered with the detection of anti-trophoblast cell antibodies; positive samples for anti-zona pellucida antibodies interfered with the detection of anti-trophoblast cell antibodies; positive samples for anti-trophoblast cell antibodies interfered with the detection of anti-ovarian antibodies and anti-human chorionic gonadotropin antibodies.

[0036] 2. Test results and interference analysis of reagent group 2 Test results (RU / mL):

[0037] Relative deviation:

[0038] Interference analysis: No interference exists.

[0039] 3. Test results and interference analysis of reagent group 3 Test results (RU / mL):

[0040] Relative deviation:

[0041] Interference analysis: Positive samples for anti-endometrial antibodies showed interference in the detection of antibodies against zona pellucida, anti-human chorionic gonadotropin (hCG), and anti-trophoblast cells; positive samples for anti-ovarian antibodies showed interference in the detection of hCG antibodies; positive samples for anti-zona pellucida antibodies showed interference in the detection of anti-ovarian antibodies and anti-trophoblast cells; positive samples for anti-trophoblast cells antibodies showed interference in the detection of anti-ovarian antibodies and anti-zona pellucida antibodies.

[0042] 4. Screening Conclusion When using antigen from kit 1 in reagent group 1 and antigen from kit 3 in reagent group 3 to test samples, there was endogenous interference, which did not meet the requirements; when using antigen from kit 2 in reagent group 2 to test samples, there was no endogenous interference, which met the requirements.

[0043] The antigen information for Set 2 is as follows:

[0044] Subsequent embodiments all used the aforementioned kit 2 antigen, which had been screened and confirmed to have no cross-interference, for reagent preparation and detection verification.

[0045] III. Detection of a composite gradient of six infertility-related autoantibodies in a sample. 1. Experimental reagents and instruments 1.1. Experimental Reagents Six types of magnetic microspheres; antisperm antibody antigen (NDX-AsAb01), anti-endometrial antibody antigen (NDX-EMAb01), anti-ovarian antibody antigen (NDX-AoAb01), anti-zona pellucida antibody antigen (NDX-AZPAb01), anti-human chorionic gonadotropin antibody antigen (NDX-AHCGAb01), and anti-trophoblast cell antibody antigen (NDX-TaAb01); secondary antibody-phycoerythrin were all purchased externally. All instrument consumables were provided by Nanjing Aituo Life Science Technology Co., Ltd. Unless otherwise specified, all other reagents and consumables were purchased externally.

[0046] 1.2. Experimental Apparatus AT2000 fully automated liquid suspension chip detector (Nanjing Aituo Life Technology Co., Ltd.), YFF-3 molecular hybridization instrument (Xinghua Analytical Instrument Factory), pipette (Thermo Fisher Scientific (China) Co., Ltd.).

[0047] 2. Experimental Methods 2.1. Preparation of the capture carrier mixture 550 μL of each of the six types of magnetic microspheres were measured into six 1.5 mL centrifuge tubes. The corresponding microsphere information was labeled on the centrifuge tubes. The microspheres were allowed to stand for 2 minutes after magnetic adsorption, and the supernatant was removed.

[0048] Add 1000 μL of 5 mM MES buffer to each centrifuge tube, magnetically adsorb and let stand for 2 minutes, then remove the supernatant.

[0049] Add 960 μL of 5 mL MMES buffer, 20 μL of 50 mg / mL NHS and 20 μL of 50 mg / mL LEDC to each centrifuge tube and mix well.

[0050] Each centrifuge tube was placed in a molecular hybridization apparatus, the rotation speed was adjusted to 28 rpm, and incubated at 37°C for 30 minutes.

[0051] After incubation, remove each centrifuge tube, magnetically adsorb and let stand for 2 minutes, then remove the supernatant.

[0052] Add 1000 μL of 5 mM MES buffer to each centrifuge tube, magnetically adsorb and let stand for 2 minutes, then remove the supernatant.

[0053] Add 725 μL of 5 mM MES buffer and 275 μL of the corresponding antigen to each centrifuge tube and mix well.

[0054] Each centrifuge tube was placed in a molecular hybridization apparatus, the rotation speed was adjusted to 28 rpm, and incubated at 37°C for 3 hours.

[0055] After incubation, remove each centrifuge tube, magnetically adsorb and let stand for 2 minutes, then remove the supernatant.

[0056] Add 1000 μL of 100 mM phosphate buffer containing 2% BSA to each centrifuge tube and mix well.

[0057] Each centrifuge tube was placed in a molecular hybridization apparatus, the rotation speed was adjusted to 28 rpm, and incubated at 37°C for 1 hour.

[0058] After incubation, remove each centrifuge tube, magnetically adsorb and let stand for 2 minutes, then remove the supernatant.

[0059] Add 1000 μL of 100 mM phosphate buffer containing 2% BSA to each centrifuge tube and mix well.

[0060] Transfer the liquid from the six centrifuge tubes to a 300mL reagent bottle, add 269mL of 100mM phosphate buffer, and mix well.

[0061] 2.2. Preparation of secondary antibody-phycoerythrin working solution Measure 2750 μL of the secondary antibody-phycoerythrin into a 300 mL reagent bottle, add 275.25 mL of 100 mM phosphate buffer, and mix well.

[0062] 2.3. Preparation of composite gradient samples of infertility-related autoantibodies Take samples close to the lower limit of the linear range and dilute them with samples close to the upper limit of the linear range, diluting them to six concentrations according to the following proportions.

[0063]

[0064] 2.4. Measurement and Analysis Place the capture vector mixture, secondary antibody-phycoerythrin working solution, and sample into the designated position on the AT2000 fully automated liquid suspension chip detector. Select six parameters related to infertility-related autoantibodies to begin the reaction. The specific reaction process is as follows: Add 25 μL of capture vector mixture, 25 μL of secondary antibody-phycoerythrin working solution and 25 μL of sample to the reaction vessel.

[0065] The reaction vessel was kept at a constant temperature and oscillated at 800 rpm for 1 hour at 37°C.

[0066] After incubation, the reaction vessel was magnetically adsorbed and allowed to stand for 1 minute before the supernatant was removed.

[0067] Add 150 μL of 100 mM phosphate buffer to each reaction well, allow it to stand for 1 minute using magnetic adsorption, and then remove the supernatant.

[0068] Add 150 μL of 100 mM phosphate buffer to each reaction vessel.

[0069] After the reaction is complete, the reaction vessel is transferred to the detection area. The instrument's light source excites all the capture carrier-analyte-secondary antibody-phycoerythrin immune complexes to generate fluorescence signals. The instrument collects the fluorescence signals, identifies the solid-phase carrier (i.e., the detection item), and analyzes the fluorescence signal of phycoerythrin to calculate the concentration of the analyte.

[0070] 3. Experimental Results The gradient sample detection results for each project are as follows: Antisperm antibodies:

[0071] Anti-endometrial antibodies:

[0072] Anti-ovarian antibodies:

[0073] Anti-zona pellucida antibodies:

[0074] Anti-human chorionic gonadotropin antibody:

[0075] Anti-trophoblast cell antibodies:

[0076] 4. Results Analysis The six infertility-related autoantibody composite gradient samples in this embodiment were tested, and all six items were correctly identified. The test results showed that the correlation coefficient r between the detected values ​​of the composite gradient samples and the theoretical values ​​of the samples was greater than 0.99, which meets the requirements of the medical diagnostic standards.

[0077] This embodiment enables rapid multiplex detection of six infertility-related autoantibodies. A single sample requires only a single loading reaction (25 μL sample volume) to obtain results for all six items. This invention saves sample volume, shortens reaction time, and improves detection efficiency.

[0078] IV. Summary of Technical Effects All raw materials used in this embodiment are commercially available and mass-produced, with no special modification requirements. The experimental design in this embodiment is simple, requiring no complex or sophisticated equipment to complete item identification and concentration detection. This embodiment demonstrates accurate identification and a simple principle in the rapid multiplex detection of different infertility-related autoantibody items. The detection and analysis process can be fully automated, requiring only a single sample addition reaction per sample, eliminating the need for multiple additions and repeated reactions. This improves the clinical application efficiency of infertility-related autoantibody detection and reduces testing costs.

[0079] Obviously, the above embodiments of the present invention are merely illustrative examples to illustrate the invention and are not intended to limit the implementation of the invention. Other obvious variations or modifications derived from the essential spirit of the invention still fall within the protection scope of the invention.

Claims

1. A method for multiplex detection of autoantibodies in infertility, characterized in that, Includes the following steps: Antigens corresponding to different infertility-related autoantibodies are coupled to different solid-phase carriers; The solid-phase support is sealed to form a capture carrier; Phycoerythrin was labeled onto the secondary antibody to form a secondary antibody-phycoerythrin complex; The analyte is mixed with the capture carrier and the secondary antibody-phycoerythrin complex to form a capture carrier-analyte-secondary antibody-phycoerythrin immune complex. The immune complex is excited by a light source to generate a corresponding fluorescence signal; The type of analyte is determined and its concentration is calculated based on the different fluorescence signals.

2. The method for multiplex detection of infertility autoantibodies according to claim 1, characterized in that, Different solid supports produce different fluorescence signals when excited by a light source, and the fluorescence signal of the phycoerythrin is related to the concentration of the analyte.

3. The method for multiplex detection of infertility autoantibodies according to claim 1, characterized in that, The solid support is a magnetic microsphere.

4. The method for multiplex detection of infertility autoantibodies according to claim 1, characterized in that, The infertility-related autoantibodies are selected from at least two of the following: antisperm antibodies, antiendometrial antibodies, antiovarian antibodies, antizona pellucida antibodies, antihuman chorionic gonadotropin antibodies, and antitrophoblast cell antibodies.

5. An antigen screening method for multiplex detection of autoantibodies in infertility, characterized in that, Includes the following steps: Multiple candidate antigen sets are provided, each containing antigens corresponding to different infertility-related autoantibodies; Multiple tests were performed on interfering samples containing a single infertility-related autoantibody using each candidate antigen kit. The relative deviation of the test results for each item within ±10% is used as the criterion for determining no interference; Antigen sets that do not interfere with the detection of any interfering samples were selected.

6. The antigen screening method for multiplex detection of autoantibodies in infertility according to claim 5, characterized in that, The infertility-related autoantibodies include antisperm antibodies, anti-endometrial antibodies, anti-ovarian antibodies, anti-zona pellucida antibodies, anti-human chorionic gonadotropin antibodies, and anti-trophoblast cell antibodies.

7. A kit for detecting autoantibodies in infertility, characterized in that, Includes the following components: Solid-phase carriers conjugated with antisperm antibodies:

1. Solid-phase carriers conjugated with anti-endometrial antibodies; 2. Solid-phase carriers conjugated with anti-ovarian antibodies; 3. Solid-phase carriers conjugated with anti-zona pellucida antibodies; 4. Solid-phase carriers conjugated with anti-human chorionic gonadotropin antibodies; 5. Solid-phase carriers conjugated with anti-trophoblast cells antibodies; 6. Secondary antibody-phycoerythrin complex.

8. The infertility autoantibody detection kit according to claim 7, characterized in that, The solid support is a magnetic microsphere.

9. The infertility autoantibody detection kit according to claim 7, characterized in that, The antigens are: antisperm antibody antigen NDX-AsAb01, anti-endometrial antibody antigen NDX-EMAb01, anti-ovarian antibody antigen NDX-AoAb01, anti-zona pellucida antibody antigen NDX-AZPAb01, anti-human chorionic gonadotropin antibody antigen NDX-AHCGAb01, and anti-trophoblast cell antibody antigen NDX-TaAb01.

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